After ovulation, the oocytes of most mammals remain blocked at the second metaphase stage of meiosis and will eventually degenerate unless sperm penetration takes place. Sperm entry activates a whole series of events in the oocyte which leads to fertilization and the development of a new individual. The earliest changes that can be recognized in the oocyte during activation are the completion of meiosis with the omission of the second polar body and the release of the cortical granules. This is followed by the formation of the male and female pronuclei containing their respective haploid sets of chromosomes. A period of DNA synthesis occurs before the two sets of chromosomes condense and come together on the mitotic spindle of the first cleavage division. Fertilization is completed with the restoration of the diploid complement of chromosomes in the nuclei of the two-cell embryo.
However, it is also known that under certain conditions, which may occur spontaneously in vivo, or in vitro, under controlled conditions, that oocytes containing DNA of all male or female origin may because activated and result in the production of an embryo. Typically such embryo does not develop into an offspring, but rather stops developing fairly early in embryogenesis.
The activation of oocytes and development of embryos that comprise DNA of all male or female origin is typically effected as a means of studying embryogenesis. For example, the activation of oocytes containing DNA of all female origin, without any contribution from the male gamete, and the production of an embryo therefrom, is known as “parthenogenesis.” This method has been used by many researchers as a means for studying embryogenesis in vitro.
Parthenogenesis is a type of gynogenesis. Gynogenesis broadly is defined as the phenomena wherein an oocyte containing all female DNA becomes activated and produces an embryo. Gynogenesis includes parthenogenesis as well as activation methods wherein the spermatozoa activates the oocyte to complete meiosis, but fails to contribute any genetic material to the resulting embryo. As in parthenogenesis, the activated oocyte does not contain DNA of male origin. However, unlike parthenogenesis, however, the male gamete does make a contribution, i.e., it stimulates oocyte activation.
Androgenesis can be considered to be the opposite of gynogenesis. This refers to the production and activation of an oocyte containing DNA of entirely male origin, and the development of an embryo therefrom.
In general, embryos that result from oocytes containing DNA of all female or male origin only develop to a certain point, and then stop developing. This is hypothesized to occur, e.g., in the case of parthenogenetic embryos of the instability of the aging unfertilized oocyte in the maintenance of the meiotic block. In fact, parthenogenetically activated oocytes may give rise to a variety of aberrations that occur during the completion of meiosis, which may result in the production of embryos of different genetic constitutions.
It is known that artificial activation of mammalian oocytes, including oocytes containing DNA of all male or female origin, can be induced by a wide variety of physical and chemical stimuli. Examples of such methods are listed in the Table below.
TABLE 1List of physical and chemical stimuli which caninduce oocyte activation in mammals.PhysicalChemicalMechanicalEnzymaticprickingtrypsin, pronase, hyaluilanidasemanipulation of oocytes in vitroOsmoticThermalIoniccoolingdivalent cationsheatingcalcium ionophoresElectricAnaestheticsgeneral - ether, ethanol, nembutal,chloroform, avertinlocal - dibucaine, tetracaine, lignocaine,procainePhenothiazine, tranquilizers thioridazine,trifluoperazine, fluphenazine,chlorpromazineProtein synthesis inhibitorscycloheximide, puromycinPhosphorylation inhibitors (e.g., DMAP)Inisitol 1,4,5-triphosphate (Ins P3)
Indeed, the activation of parthenogenetic oocytes has been well reported in the literature. For example, Ware et al, Gamete Research, 22:265-275 (1989) teach the ability of bovine oocytes to undergo parthenogenetic activation using Ca++, Mg++—H+ ionophore (A23187) or electric shock. Also, Yang et al, Soc. Study Reprod., 46:117 (1992) teaches activation of bovine follicular oocytes using cycloheximide and electric pulse treatment. Graham C. F. in Biol. Rev., 49:399-422 (1979) describes then existing methods for activating parthenogenetic mammalian embryos. Further, Matthew H. Kaufman, in Prog. in Anat, Vol. 1:1-34, ed. R. G. Harrison and R. L. Holmes, Cambridge Press, London, UK (1981) reviews parthenogenesis and methods of activation. The parthenogenetic activation of rabbit and mouse oocytes is also disclosed by Ozil, Jean Pierre, Devel., 109:117-127 (1990); Kubiak, Jacek, Devel. Biol., 136:537-545 (1989); Onodera et al, Gamete Research, 22:277-283 (1989); Siracusa et al, J. Embryol. Exp. Morphol., 43:157-166 (1978); and Szollosi et al, Chromosoma, 100:339-354 (1991). Still further, the activation of unfertilized sea urchin eggs is disclosed by Steinhardt et al, Nature, 252:41-43 (1974); and Whitaker, M., Nature, 342:636-639 (1984). Also, the parthenogenetic activation of human oocytes has been reported. (See, e.g., De Sutter et al, J. Associated Reprod. Genet., 9(4):328-336 (1992).)
In general, the goal of such artificial oocyte activation methods has been known in biological research, in particular the study of embryonic development, and the mechanisms which are involved in oocyte activation.
In recent years, a significant goal of many research groups has been the identification of methods that efficiently and reproducibly give rise to pluripotent or embryonic stem cells (ES cells), and ES or pluripotent cell lines. ES cells are extremely desirable because of their pluripotency which allows them to give rise to any differentiated cell type. Also, ES cells are useful for the production of chimeric animals and as an in vitro model for differentiation studies, especially the study of genes involved in early development.
Methods for deriving embryonic stem (ES) cell lines in vitro from early preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol., 121:1-9 (1987)) is present.
ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early development. Mouse ES cells can give rise to germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309:255-256 (1984)).
In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al., Biol. Reprod, 40:1027-1035 (1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei from mouse embryos which beyond the eight-cell stage after transfer reportedly do not support the development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958 (1993)). Therefore, ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures.
Some research groups have reported the isolation of purportedly pluripotent embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43:255260 (1991), reports the establishment of purportedly stable, pluripotent cell lines from pig and sheep blastocysts which exhibit some morphological and growth characteristics similar to that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:51-56 (1990) discloses maintenance and differentiation in culture of putative pluripotential embryonic cell lines from pig blastocysts. Gerfen et al., Anim. Biotech, 6(1):1-14 (1995) discloses the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned medium, and reportedly differentiate into several different cell types during culture.
Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992) reports cultured, bovine embryonic stem cell-like cell lines which survived three passages, but were lost after the fourth passage. Handyside et al., Roux's Arch. Dev. Biol., 196:185-190 (1987) discloses culturing of immunosurgically isolated inner cell masses of sheep embryos under conditions which allow for the isolation of mouse ES cell lines derived from mouse ICMs. Handyside et al. reports that under such conditions, the sheep ICMs attach, spread, and develop areas of both ES cell-like and endoderm-like cells, but that after prolonged culture only endoderm-like cells are evident.
Cherny et al., Theriogenology, 41:175 (1994) reported purportedly pluripotent bovine primordial germ cell-derived cell lines maintained in long-term culture. These cells, after approximately seven days in culture, produced ES-like colonies which stained positive for alkaline phosphatase (AP), exhibited the ability to form embryoid bodies, and spontaneously differentiated into at least two different cell types. These cells also reportedly expressed mRNA for the transcription factors OCT4, OCT6 and HES 1, a pattern of homeobox genes which is believed to be expressed by ES cells exclusively.
Campbell et al., Nature, 380:64-68 (1996) reported the production of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day nine ovine embryos cultured under conditions which promote the isolation of ES cell lines in the mouse. The authors concluded that ED cells from day nine ovine embryos are totipotent by nuclear transfer and that totipotency is maintained in culture.
Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported the isolation and characterization of purportedly permanent cell lines from inner cell mass cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or 9 day bovine blastocysts under different conditions to determine which feeder cells and culture media are most efficient in supporting the attachment and outgrowth of bovine ICM cells. They concluded that the attachment and outgrowth of cultured ICM cells is enhanced by the use of STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial cells) and by the use of charcoal-stripped serum (rather than normal serum) to supplement the culture medium. Van Stekelenburg et al reported, however, that their cell lines resembled epithelial cells more than pluripotent ICM cells.
Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et al, WO 90/03432, published Apr. 5, 1990, and Wheeler et al, WO 94/26889, published Nov. 24, 1994, report the isolation, selection and propagation of animal stem cells which purportedly may be used to obtain transgenic animals. Evans et al. also reported the derivation of purportedly pluripotent embryonic stem cells from porcine and bovine species which assertedly are useful for the production of transgenic animals. Further, Wheeler et al, WO 94/26884, published Nov. 24, 1994, disclosed purported embryonic stem cells which are assertedly useful for the manufacture of chimeric and transgenic ungulates. However, to the best of the inventors' knowledge, this work has never been reported in any peer-reviewed journal.
Quite recently, two research groups simultaneously reported the production of purified non-human primate and human ES cells. These ES cell lines were derived from non-human primate and human embryos. These purported ES cell lines were reported to be SSEA-1 negative, SSEA-4, and SEA-3 positive, TRA-1-60 and TR-A1-81 positive, and alkaline phosphatase positive, to develop into all three embryonic germ layers (endoderm, mesoderm, ectoderm), to maintain a normal karyotype even after prolonged culturing, and to proliferate indefinitely in vitro in an undifferentiated state.
Also recently, a group of scientists at the University of Massachusetts and Advanced Cell Technology reported the production of a “human embryo” by cross species nuclear transplantation of human differentiated cells into a bovine oocyte, and the potential use thereof for the production of human ES cells. However, notwithstanding what has been reported, improved methods for obtaining ES cells, and methods for maintaining such cells in vitro for indefinite periods would be extremely useful.